The technology of PCR permits amplification and subsequent detection of minute quantities of a target nucleic acid. Details of PCR are well described in the art, including, for example, U.S. Pat. Nos. 4,683,195 to Mullis et al., 4,683,202 to Mullis and 4,965,188 to Mullis et al. Generally, oligonucleotide primers are annealed to the denatured strands of a target nucleic acid, and primer extension products are formed by the polymerization of deoxynucleoside triphosphates by a polymerase. A typical PCR method involves repetitive cycles of template nucleic acid denaturation, primer annealing and extension of the annealed primers by the action of a thermostable polymerase. The process results in exponential amplification of the target nucleic acid, and thus allows the detection of targets existing in very low concentrations in a sample.
PCR is widely used in a variety of applications, including biotechnological research, clinical diagnostics and forensics. However, the methodology is subject to practical limitations that result in less than optimal efficiency and specificity. In particular, before the first cycle of a PCR experiment (i.e. at "zero cycle"), the reagents for amplification are typically mixed and stored at room temperature or lower. Because thermostable polymerases, for example, Thermus aquaticus (Taq) polymerase, have residual activity even at 0.degree. C., relatively large quantities of non-specific products can be formed by low stringency priming and replication. The non-specific products, known as zero cycle artifacts, include primer-dimers formed by ligation of primers having homology at their 3' ends. Because of the micromolar concentrations of primers used in PCR relative to the often minute concentrations of target, the formation of primer-dimers is predominant. Primer-dimers are thus particularly pervasive zero-cycle artifacts. Other primer based amplification systems, such as solid phase amplification, similarly suffer from primer-dimer artifacts.
The formation of zero-cycle artifacts during amplification has practical consequences. Reagents, including primers and deoxyribonucleosides, may be depleted, and the non-specific side products act as competitive inhibitors with respect to the target for the polymerase and other limiting components of the reaction. Consequently, amplification efficiency may be decreased and assay precision degraded. Any decrease in amplification efficiency may adversely effect the assay detection limit, and thus potentially cause false negative results. As demonstrated in accordance with the present invention, primer-dimer formation can reduce efficiency of target amplification to such a degree that the amplified product is not detectable on a stained gel. Such a result would clearly be undesirable in tests for pathogenic organisms, such as HIV.
Specificity is particularly important in homogeneous PCR reactions. See, e.g., EPA 487218 to Mitoma; EPA 512334 to Higuchi. In the homogeneous assays, PCR amplification and detection are coupled by contacting the reaction mixture, during or after amplification, with a fluorescent pigment that undergoes a detectable change in fluorescence upon reaction with a double-stranded nucleic acid. For example, when PCR is conducted in the presence of ethidium bromide, the production of double-stranded DNA is correlated with an increase in fluorescence as free ethidium bromide becomes intercalated into double-stranded DNA. Generally, amplification and detection are carried out in the same vessel. Changes in fluorescence are detected spectrophotometrically, and thus detection requires neither separation of PCR products nor hybridization. Because detection is based upon formation of double-stranded DNA generally, and fails to discriminate between target DNA and non-specific products, the formation of double-stranded artifacts such as primer-dimers is fatal to the specificity of the homogeneous assay.
Various strategies have been developed to increase PCR specificity. Theoretically, primer-dimer artifacts can be avoided by selecting primers with no 3'-homology. In practice, however, some 3' homology may be unavoidable, particularly in applications that require mixtures of primers. Coamplification of numerous strains or alleles of a target are typical applications that require a large number of primers.
Specificity can also be improved by increasing stringency, for example, by increasing the annealing temperature or incorporating denaturing solvents. However, increasing stringency may lead to false negative results because the assay's ability to detect mutated forms of the target, which may have been amplified at lower stringency, is reduced.
In another method of reducing zero-cycle artifacts, the so-called "hot start" method of PCR, the reaction is started by the addition of polymerase to hot reagent mixtures. (See, e.g., Erlich et al. (1991) Science 252:1643.) Primer-dimers are reduced since the reactive intermediates formed by cross-reaction of primers are thermally unstable. However, this method does not provide the convenience of room temperature preparation, and is subject to complications caused by timing errors resulting from manual addition of polymerase to multiple (typically 96) PCR tubes.
Thermolabile physical barriers, such as paraffin beads or overlays, have been used to physically separate one or more PCR components from the others until temperatures suitable for high stringency priming are reached (See, e.g., Hebert et al. (1993) Molecular and Cellular Probes 7:249). However, these methods are generally inconvenient and require considerable manual dexterity.
Thermally labile antibodies to Taq polymerase have been used to inhibit Taq polymerase at low temperatures in an attempt to limit zero cycle artifacts. (See, e.g., Sharkey et al. (1994) Bio/Technology 12:506; U.S. Pat. No. 5,338,671 to Scalice et al.) When the temperature is elevated in the PCR thermal cycling, the antibodies are thermally denatured and active polymerase is released. However, even avid antibodies do not completely inhibit polymerase activity. For example, one micromolar antibody having affinity of 10.sup.10 M.sup.-1 acting on polymerase at a concentration of 10 nanomalar in a volume of 100 microliters would leave 60 million molecules of free active polymerase at equilibrium. Since primer levels used in PCR are relatively large, sizable numbers of primer-dimer intermediates can nonetheless be formed and amplified. As a result and as demonstrated in accordance with the present invention, anti-Taq antibody alone may be insufficient to suppress primer-dimer formation, especially in cases in which the primers have substantial 3' homology or in which the homology consists of strong G--C bonds.
Enzymes capable of digesting primer-dimer intermediates have also been disclosed for use in side product suppression. Zhu et al. (1991) Nucleic Acids Res. 19:251) report the use of exonuclease III (Exo III) for "pre-PCR sterilization" to reduce amplicon and primer-dimer carry over. However, since Exo III catalyzes the sequential cleavage of 5' mononucleotides from the 3' hydroxyl end of duplex DNA, it may also attack target DNA. Further Zhu et al. report that Exo III does not degrade single-stranded DNA, and thus single-stranded primer-dimers could be expected to escape Exo III treatment and thereby be susceptible to amplification. Muralidhar et al. (1992) Gene 117:107 report the use of T7 exonuclease to reduce amplification of carry over PCR product molecules. The contaminating PCR molecules are preferentially inactivated due to their symmetric geometry relative to genomic target molecules. Muralidhar et al. fail to reduce primer-dimer products, and note that the geometry of primer-dimers has not been established.
The enzyme uracil-N-glycosylase (UNG) has also been used in a preamplification step to cleave products made during the zero cycle at incorporated uracil residues. (See, e.g., Longo et al. (1990) Gene 93:125; Espy et al. (1993) J. Clin. Microbiol. 31:2361.) Deoxyuridine triphosphate (dUTP) is substituted for deoxythymidine triphosphate (dTTP) in the PCR and thus PCR products can be distinguished from template DNA. The enzyme UNG is included in the premix, and catalyzes the excision of uracil from single or double-stranded DNA present in the reaction prior to the first PCR cycle. The resulting abasic polynucleotides are susceptible to hydrolysis and cannot function as templates during PCR. While UNG is reportedly inactivated by thermal denaturation, residual activity may degrade amplification products synthesized during PCR. Further, Longo et al. compared the relative amount of amplification product in the presence and absence of UNG treatment, and reported a reduction in the intensity of the amplified target in reactions with UNG treatment. Thus UNG treatment would not be expected to solve the problem of inefficiency of product amplification.
Epsy et al. report that the efficiency of UNG in inactivating amplified DNA is dependent upon the length of the DNA. In particular, UNG was ineffective in inactivating PCR amplicons of less than 100 base pairs. Accordingly, UNG fails to inactivate primer-dimers.
As demonstrated in accordance with the present invention, neither Exo III nor UNG is particularly effective for suppression of primer-dimers and improved amplification efficiency. Further, as discussed hereinabove, the prior art methods of suppressing zero cycle artifacts suffer from practical deficiencies. Accordingly, there is a need in the art for practical and effective methods of suppressing zero cycle artifacts and thus increasing PCR efficiency and specificity.